重组福安泰-03抑制人脐静脉血管内皮细胞的迁移和增殖

研究重组福安泰-03(recombinant Fuantai-03,rFAT-03)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)迁移和增殖的影响.聚碳酸酯膜小室趋化运动模型(transwell model)检测rFAT-03对HUVECs迁移能力的影响;MTT法检测rFAT-03对HUVECs和多种人癌细胞生长的影响;荧光显微镜观察rFAT-03作用下HUVECs的形态变化;膜联蛋白V-异硫氰酸荧光素(annexin V-fluorescein isothiocyanate,annexinV-FITC)双染检测rFAT-03对HUVECs早期凋亡的影响;流式细胞术分析rFAT-03对HUVECs周期及凋亡的影响;Western印迹检查rFAT-03对HUVECs血管内皮细胞生长因子(VEGF)、Bax和Bcl-2表达的影响.结果显示,rFAT-03明显抑制HUVECs细胞的迁移和增殖,其抑制效果与剂量和作用时间相关.0.20mg/mL恩度(endostar),0.10、0.20mg/mLrFAT-03作用HUVECs24h,对细胞迁移的抑制率分别为32.0%、32.6%、57.1%(P0.01).0.20mg/mL恩度,0.05、0.10、0.20mg/mLrFAT-03作用HUVECs72h,其对细胞生长的抑制率分别为40.9%、63.7%、69.3%、87.0%.但rFAT-03对多种人癌细胞株的生长却无明显影响.rFAT-03处理HUVECs24h,细胞的早期凋亡率增加(P0.05).rFAT-03阻滞HUVECs于G0/G1期,并呈现典型的凋亡峰.终浓度为0.05、0.10、0.20mg/mLrFAT-03作用于HUVECs24h,G0/G1期细胞指数分别为63.4%、67.5%和75.7%(对照组为62.1%),凋亡指数分别为4.2%、8.5%和10.3%.rFAT-03下调HUVECs的VEGF和抑调亡基因Bcl-2的表达,上调促凋亡基因Bax的表达,其效果与剂量相关.结果提示,rFAT-03明显抑制HUVECs细胞的迁移和增殖,诱导其凋亡,它的这些作用与其下调VEGF、Bcl-2表达,上调Bax表达密切相关.

Recombinant Fuantai-03 Inhibits the Migration and Proliferation in Human Umbilical Vein Endothelial Cells

The present study was conducted to investigate the effects of recombinant Fuantai-03 (rFAT- 03）on the migration and proliferation in human umbilical vein endothelial cells (HUVECs). Migration assay was performed using a Transwell model with polycarbonate membrane; cell growth inhibitory effect was measured by MTT assay; apoptotic induction of HUVECs by rFAT-03 was determined by fluorescence microscopy and flow cytometry; and Western blot analysis was performed for examining expressions of vascular endothelial growth factor (VEGF), Bcl-2 and bax. rFAT-03 obviously inhibited the migration and proliferation of HUVECs in a dose- and time-dependent manner. The doses 0.20 mg/mL endostar, 0.10 and 0.20 mg/mLrFAT-03 decreased the percentage of migrating HUVECs by 32.0%，32.6%, and 57.1%（P<0.01）, respectively. The doses 0.20 mg/mL endostar and 0.05, 0.10, 0.20 mg/mL rFAT-03 displayed growth inhibitory activity against HUVECs with inhibition rates of 40.9%, 63.7%, 69.3% and 87.0% for 72 hours, respectively, but did not show significant effects on several human malignant tumor cell lines. rFAT-03-treated HUVECs showed typical morphologic and cellular evidence of apoptosis. The expressions of VEGF and Bcl2 in the rFAT-03-treated HUVECs were evidently down-regulated, and expression of bax was obviously up-regulated. These findings provide evidence that rFAT-03 significantly inhibits the migration and proliferation of HUVECs and induces apoptosis, and the effects of rFAT03 might result from the down-regulation of expressions of VEGF and Bcl-2 and up-regulation of expression of bax in the rFAT-03-treated HUVECs.